ATR FTIR Investigation of Beta-Lactamase Catalysis

9730035 Fink拟议的研究涉及两个相关领域：（1）使用傅立叶变换红外（FTIR）光谱，在衰减全反射（ATR）模式下，研究酶-底物中间体和酶-抑制剂复合物，（2）研究β-内酰胺酶的催化和抑制，特别是使用定点诱变和结构探针的组合，包括ATR FTIR。 β-内酰胺酶是青霉素类抗生素耐药的主要来源。尽管有相当多的研究，但对其催化机制尚未达成共识。一个详细的动力学和结构表征选定的活性位点突变体的计划，以回答一些具体的问题，关于β-内酰胺酶催化机制。红外光谱可以揭示有关催化机制的关键信息，以及结合抑制剂和药物的构象和相互作用。通过将酶固定在ATR流动池的内反射元件上，可以获得稳定的酶-底物或酶-抑制剂复合物的IR光谱。ATR FTIR研究将首先应用于β-内酰胺酶的复合物，随后扩展到其他酶系统。 β-内酰胺酶是由许多细菌产生的酶，是青霉素耐药性的主要来源。计划中的研究旨在了解这些酶如何工作，以及它们如何被抑制。一个详细的动力学和结构表征选定的活性位点突变体将进行回答一些具体的问题，有关的催化机制。所学到的信息将有助于理解β-内酰胺酶如何工作和潜在抑制剂的设计。红外光谱可以揭示催化过程中键变化的关键信息。将开发一种新的方法来研究酶-底物中间体和酶-抑制剂复合物，特别是β-内酰胺酶的中间体和酶-抑制剂复合物。

9730035 Fink The proposed research involves two related areas: (1) the use of Fourier Transform Infrared (FTIR) spectroscopy, in the attenuated total reflectance (ATR) mode, to study enzyme-substrate intermediates and enzyme-inhibitor complexes, and (2) studies on beta-lactamase catalysis and inhibition, especially using a combination of site-directed mutagenesis and structural probes, including ATR FTIR. Beta-lactamases are the major source of resistance to penicillin antibiotic therapy. In spite of considerable study there is no consensus on their catalytic mechanism. A detailed kinetic and structural characterization of selected active-site mutants is planned to answer a number of specific questions regarding the beta-lactamase catalytic mechanism. Infrared spectroscopy can reveal critical information about catalytic mechanisms, as well as the conformation and interactions of bound inhibitors and drugs. By immobilizing the enzyme to the internal reflectance element of an ATR flow cell it is possible to acquire the IR spectra of stabilized enzyme-substrate or enzyme-inhibitor complexes. The ATR FTIR studies will initially be applied to complexes of beta-lactamase, and subsequently extended to other enzyme systems. Beta-lactamases are enzymes made by many bacteria and are the major source of resistance to penicillin. The planned research is aimed at understanding how these enzymes work, and how they may be inhibited. A detailed kinetic and structural characterization of selected active-site mutants will be undertaken to answer a number of specific questions regarding the catalytic mechanism. The information learned will contribute to understanding how beta-lactamases work and to the design of potential inhibitors. Infrared spectroscopy can reveal critical information about bond changes during catalysis. A new procedure will be developed to study enzyme-substrate intermediates and enzyme-inhibitor complexes, especially those of beta-lactamase.